Carbon-substituted diketopiperazine delivery systems

ABSTRACT

Compositions useful in the delivery of active agents are provided. These delivery compositions include (a) an active agent; and (b) a carrier of at least one mono-C-substituted or di-C-substituted diketopiperazine. Methods for preparing these compositions and administering these compositions are also provided.

FIELD OF THE INVENTION

The present invention relates to compositions for delivering activeagents, and particularly biologically active agents. The carriers inthese compositions facilitate the delivery of a cargo to a target. Thesedelivery compositions are particularly useful in the oral delivery ofbiologically active agents such as pharmacologically or therapeuticallyactive agents. Methods for the preparation and for the administration ofsuch compositions are also disclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering active agents are often severelylimited by biological, chemical, and physical barriers. Typically, thesebarriers are imposed by the environment through which delivery occurs,the environment of the target for delivery, or the target itself.

Biologically active agents are particularly vulnerable to such barriers.For example in the delivery to animals of pharmacological andtherapeutic agents, barriers are imposed by the body. Examples ofphysical barriers are the skin and various organ membranes that must betraversed before reaching a target. Chemical barriers include, but arenot limited to, pH variations, lipid bi-layers, and degrading enzymes.

These barriers are of particular significance in the design of oraldelivery systems. Oral delivery of many biologically active agents wouldbe the route of choice for administration to animals if not forbiological, chemical, and physical barriers such as varying pH in thegastrointestinal (GI) tract, powerful digestive enzymes, and activeagent impermeable gastrointestinal membranes. Among the numerous agentswhich are not typically amenable to oral administration are biologicallyactive peptides, such as calcitonin and insulin; polysaccharides, and inparticular mucopolysaccharides including, but not limited to, heparin;heparinoids; antibiotics; and other organic substances. These agents arerapidly rendered ineffective or are destroyed in the gastrointestinaltract by acid hydrolysis, enzymes, or the like.

Earlier methods for orally administering vulnerable pharmacologicalagents have relied on the co-administration of adjuvants (e.g.,resorcinols and non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation.

Liposomes have also been described as drug delivery systems for insulinand heparin. See, for example, U.S. Pat. No. 4,239,754; Patel et al.(1976), FEBS Letters, Vol. 62, pg. 60; and Hashimoto et al. (1979),Endocrinology Japan, Vol. 26, pg. 337.

However, broad spectrum use of such drug delivery systems is precludedbecause: (1) the systems require toxic amounts of adjuvants orinhibitors; (2) suitable low molecular weight cargos, i.e. activeagents, are not available; (3) the systems exhibit poor stability andinadequate shelf life; (4) the systems are difficult to manufacture; (5)the systems fail to protect the active agent (cargo); (6) the systemsadversely alter the active agent; or (7) the systems fail to allow orpromote absorption of the active agent.

More recently, microspheres of artificial polymers of mixed amino acids(proteinoids) have been used to deliver pharmaceuticals. For example,U.S. Pat. No. 4,925,673 describes drug-containing proteinoid microspherecarriers as well as methods for their preparation and use. Theseproteinoid microspheres are useful for the delivery of a number ofactive agents.

There is still a need in the art for simple, inexpensive deliverysystems which are easily prepared and which can delivery a broad rangeof active agents.

SUMMARY OF THE INVENTION

Compositions useful in the delivery of active agents are provided. Thesedelivery compositions comprise (a) an active agent; and (b) a carriercomprising at least one mono-C-substituted or di-C-substituteddiketopiperazine. Biologically active agents and pharmacologicallyactive agents may be incorporated as the active agent, and thesecompositions may be in the form of microspheres.

Also contemplated is a method for preparing these compositions whereinat least one active agent is mixed with a carrier as described above orwherein the carrier is solubilized in a solvent, and the carriersolution is contacted with the active agent and a precipitator solutionin which the carrier is insoluble.

In a further embodiment, the compositions are administered, preferablyorally, to animals.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an illustration of a reaction scheme for the preparation ofdiketopiperazines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is suited to the delivery of any active agentsthrough various biological, chemical, and physical barriers. It isparticularly suited to the delivery of active agents which are subjectto environmental degradation. Other advantages provided by the presentinvention include the use of readily available or easy to prepare,inexpensive starting materials. The formulation methods of the presentinvention are cost-effective for preparing and isolating thesecompositions, are simple to perform, and are amenable to industrialscale up for commercial production.

Active Agents

Active agents suitable for use in the present invention includebiologically active agents, chemically active agents, including, but notlimited to, fragrances, as well as other active agents such as, forexample, cosmetics.

Biologically active agents include, but are not limited to, pesticides,pharmacological agents, and therapeutic agents. For example,biologically active agents suitable for use in the present inventioninclude, but are not limited to, peptides, and particularly smallpeptides; hormones, and particularly hormones which by themselves do notor only pass slowly through the gastrointestinal mucosa and/or aresusceptible to chemical cleavage by acids and enzymes in thegastrointestinal tract; polysaccharides, and particularly mixtures ofmuco-polysaccharides; carbohydrates; lipids; or any combination thereof.Further examples include, but are not limited to, human growth hormones;bovine growth hormones; growth releasing hormones; interferons;interleukin-1; insulin; heparin, and particularly low molecular weightheparin; calcitonin; erythropoietin; atrial naturetic factor; antigens;monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropinreleasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium ordisodium chromoglycate); vancomycin; desferrioxamine (DFO);anti-microbials, including, but not limited to anti-fungal agents; orany combination thereof.

The compositions of the present invention may include one or more activeagents.

Diketopiperazines

The diketopiperazines of the present invention are six member ringcompounds. The ring includes two nitrogen atoms and is substituted attwo carbons with two oxygen atoms. Preferably, the carbonyl groups areat the 1 and 4 ring positions. These rings are further substituted ateither or both of the other two carbon atoms of the ring.

Most preferred diketopiperazines are compounds of the formula:

wherein R and R¹ independently are hydrogen, C₁-C₂₄ alkyl, C₁-C₂₄alkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl)phenyl, (C₁-C₁₀ alkenyl)phenyl,(C₁-C₁₀ alkyl)naphthyl, (C₁-C₁₀ alkenyl)naphthyl, phenyl (C₁-C₁₀ alkyl),phenyl(C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), and naphthyl (C₁-C₁₀alkenyl); but both R and R¹ can not be hydrogen; either or both R or R¹independently may optionally be substituted with C₁-C₄ alkyl, C₁-C₄alkenyl, C₁-C₄ alkoxy, —OH, —SH, and —CO₂R² or any combination thereof;R² is hydrogen, C₁-C₄ alkyl or C₁-C₄ alkenyl; and either or both R andR¹ independently may optionally be interrupted by oxygen, nitrogen,sulfur, or any combination thereof.

The phenyl or naphthyl groups may optionally be substituted. Suitable,but non-limiting, examples of substituents are C₁-C₆ alkyl, C₁-C₆alkenyl, C₁-C₆ alkoxy, —OH, —SH, or CO₂R³ wherein R³ is hydrogen, C₁-C₆alkyl, or C₁-C₆ alkenyl. When one of R or R¹ is hydrogen, thediketopiperazine is mono-carbon-(C)-substituted. When neither R nor R¹is hydrogen, the diketopiperazine is di-carbon-(C)-substituted.

Preferably, R, R¹, or both R and R¹, contain at least one functionalgroup, a functional group being a non-hydrocarbon portion responsiblefor characteristic reactions of the molecule. Simple functional groupsare heteroatoms including, but not limited to halogens, oxygen, sulfur,nitrogen, and the like, attached to, the carbon of an alkyl group by asingle or multiple bond. Other functional groups include, but are notlimited to, for example, hydroxyl groups, carboxyl groups, amide groups,amine groups, substituted amine groups, and the like.

Preferred diketopiperazines are those which are substituted at one ortwo of the carbons of the ring with a functional group that includes atleast one carboxyl functionality.

Diketopiperazines typically are formed from α-amino acids. The “term”amino acid used with respect to diketopiperazines also includes anycarboxylic acid having at least one free α-amine group and includesnaturally occurring and synthetic α-amino acids and all optical isomersthereof. Preferably, the diketopiperazines are formed from two aminoacids which are the same or optical isomers of one another. Typicalamino acids useful in the preparation of diketopiperazines are naturalor synthetic amino acids having the formula:HN(R⁴)—(R⁵)—OH  II

-   -   R⁴ is hydrogen, C₁ to C₂₄ alkyl, C₁-C₂₄ alkenyl, phenyl,        naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₁-C₁₀ alkenyl) phenyl,        (C₁-C₁₀ alkyl) naphthyl, (C₁-C₁₀ alkenyl) naphthyl, phenyl        (C₁-C₁₀ alkyl), phenyl (C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀        alkyl), and naphthyl (C₁-C₁₀ alkenyl); optionally R⁴ may be        substituted with C₁-C₄ alkyl, C₁-C₄ alkenyl, C₁-C₄ alkoxy, —OH,        —SH, —CO₂R⁶, or any combination thereof; R⁶ is hydrogen C₁-C₄        alkyl, or C₁-C₄ alkenyl; and R⁴ may optionally be interrupted by        oxygen, nitrogen, sulfur, or any combination thereof.

The phenyl or naphthyl groups may optionally be substituted. Suitable,but non-limiting examples of substituents are C₁-C₆ alkoxy, —OH, —SH, orCO₂R⁷, wherein R⁷ is hydrogen C₁-C₆ alkyl, or C₁-C₆ alkenyl.

R⁵ has the formula

wherein R⁸ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁to C₁₀ alkyl)-phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀alkyl)naphthyl, (C₁ to C₁₀ alkenyl)naphthyl, phenyl (C₁ to C₁₀ alkyl),phenyl (C₁ to C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), and naphthyl(C₁-C₁₀ alkenyl); R⁸ may optionally be substituted with C₁ to C₂₄ alkylor C₁ to C₂₄ alkenyl; and R⁵ may optionally be interrupted by oxygen,nitrogen, sulfur or any combination thereof.

The phenyl or naphthyl groups may optionally be substituted. Suitablebut non-limiting examples of substituents are C₁ to C₆ alkyl, C₁ to C₆alkenyl, C₁-C₆ alkoxy, hydroxy, thio, or CO₂R⁹ alkenyl, wherein R⁹ ishydrogen, C₁-C₆ alkyl, or C₁-C₆ alkenyl.

The preferred naturally occurring amino acids for preparation of thediketopiperazines of the present invention are alanine, arginine,asparagine, aspartic acid, citrulline, cysteine, cystine, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,or hydroxy proline.

The preferred non-naturally occurring amino acids for use herein arephenylglycine, α-aminobutyric acid, α-amino isobutyric acid,aminobenzoic acid, aminohippuric acid, and cysteine.

Preferably, the α-amino acids of which the diketopiperazines are derivedare glutamic acid, aspartic acid, tyrosine, phenylalanine, and opticalisomers of any of the foregoing. Most preferably, the diketopiperazinesuseful in the present invention are prepared from trifunctional aminoacids such as L-glutamic acid and L-aspartic acid which cyclize to formdiketopiperazines.

Dipiperazine ring systems may be generated during thermal polymerizationor condensation of amino acids or amino acid derivatives. (Gyore, J;Ecet M. Proceedings Fourth ICTA (Thermal Analysis), 1974, 2, 387-394(1974)). These six membered ring systems were presumably generated byintra-molecular cyclization of the dimer prior to further chain growthor directly from a linear peptide (Reddy, A. V., Int. J. Peptide ProteinRes., 40, 472-476 (1992); Mazurov, A. A. et al., Int. J. Peptide ProteinRes., 42, 14-19 (1993)).

Diketopiperazines can also be formed by cyclodimerization of amino acidester derivatives as described by Katchalski et al., J. Amer. Chem.Soc., 68, 879-880 (1946), by cyclization of dipeptide ester derivatives,or by thermal dehydration of amino acid derivatives and high boilingsolvents as described by Kopple et al., J. Org. Chem., 33 (2), 862-864(1968).

A typical synthesis of a diketopiperazine is illustrated in FIG. 1. TheCOOH group(s) of an amino acid benzyl ester are activated in step 1 toyield a protected ester. The amine is deprotected and cyclized viadimerization in step, 2, providing a diketopiperazine di-ester. Finallyin step 3, the COOH group(s) are deprotected to provide thediketopiperazine.

Ester derivatives of these diketopiperazine carriers are also useful inthe present invention.

Delivery Systems

The carriers of the present invention are pharmacologically harmless, asare the microspheres prepared therefrom. They do not effectively impairthe active (i.e. biological, chemical, therapeutical, pharmacological,or the like) agent.

The diketopiperazine carriers of the present invention may be used toprepare compositions for delivering active agent cargoes, andparticularly biologically active agent cargoes. Delivery compositionswhich include the active agent and the carrier may be in the form ofmixtures of active agent and carrier or the carrier may form amicrosphere which contains the active agent. The carrier describedherein facilitates the delivery of the cargo to a target.

Microspheres containing an active agent can generally be of the matrixform or the microcapsule form. The matrix form includes both a hollowmatrix sphere in which the carrier forms a matrix shell around a hollowcenter and the active agent is distributed throughout the matrix and asolid matrix sphere in which the carrier forms a spherical matrixcontinuum in which the active agent is distributed.

The microcapsule form is one in which the encapsulated active agenteither is in solution or is a solid, with the carrier forming a shellaround the encapsulated material. The microcapsule form is the form mostoften taken by the self assembly of the carriers of the presentinvention.

Delivery compositions may be mixtures which may be formulated simply bymixing the carrier with the active agent prior to administration. If thedelivery composition is to be of the microsphere form, carriermicrospheres can be prepared by dissolving the carrier in an appropriatesolute and then stimulating self assembly by contacting the carriersolution with a precipitator. Solubility of the carrier can be regulatedby the selection of the appropriate diketopiperazine.

Furthermore, the diketopiperazines, and therefore, the compositions ofthe present invention can be pH adapted to be selectively soluble inspecific acidic, basic, or neutral pH ranges.

Delivery compositions which are targeted to an acidic environment can bemade selectively soluble at acidic pH, such as the pH in the stomach.These compositions are prepared with an acid-soluble carrier. Theacid-soluble carrier exists largely in the cation form in at least aportion of the pH range from about 1 to about 6.8. However, above about6.8 or at selected ranges above pH 6.8, the carrier is largelyunprotonated and insoluble in water. Therefore, the carrier could selfassemble to miciospheres at basic or neutral pH, and the active agent inthe delivery composition would not be released until the carriersolubilizes upon encountering an acidic pH.

Delivery compositions which are to be targeted to an alkalineenvironment can be made selectively soluble at alkaline pH, such as thepH in the distal portion of the intestine. These compositions areprepared with a base-soluble carrier. The base-soluble carrier existslargely in an anionic form in at least a portion of the pH range of fromabout 7.2 to about 11. However, below and at pH 7.2, the carrier islargely protonated and insoluble in water. Therefore, the carrier couldself assemble to microspheres at acidic or neutral pH, and the activeagent in the delivery composition would not be released until thecarrier solubilizes upon encountering a basic pH.

Delivery compositions which are targeted to a neutral environment can bemade selectively soluble at neutral pH. These compositions are preparedwith a neutral-soluble carrier. The neutral-soluble carrier existslargely in a neutral form at neutral pH, i,e. from about 6.8 to about7.2. However, above or below this range, the carrier is insoluble inwater. Therefore, the carrier could self assemble to microsplieres atacidic or basic pH, and the active agent in the delivery compositionwould not be released until the carrier solubilizes upon encountering aneutral pH.

In a typical formulation, the final solution can contain from about 10mg to about 2000 mg of carrier per ml of solution, preferably betweenabout 75 to about 500 mg of carrier per ml of solution, and mostpreferably from about 75 to about 200 mg per ml. Optionally, the mixtureis heated to a temperature between about 20° C. and about 60° C.,preferably about 40° C., until the carrier dissolves. Particulatesremaining in the solution may be filtered out by conventional means suchas gravity filtration over filter paper. The carrier solution usually ismaintained at the elevated temperature and is mixed with the activeagent and a precipitator, for example, an acid solution such as, forexample, aqueous acetic or citric acid at a concentration ranging fromabout 1N to about 3N for acid insoluble carriers, a basic solution forbase insoluble carriers, and a neutralizing solution for neutralinsoluble carriers. The active agent can be mixed with the precipitatingsolution or can be used separately. The resultant mixture is maintainedfor a period of time sufficient for microsphere formation as observed bylight microscopy. Although it is preferred that the precipitatingsolution is added to the carrier solution, the carrier solution can beadded to the precipitating solution as well.

The solutions above may optionally contain additives such as stabilizingadditives. The presence of such additives promotes the stability anddispersability of the active agent in solution. The stabilizingadditives may be employed at a concentration ranging between about 0.1and 5% (w/v), preferably about 0.5% (w/v). Suitable, but non-limitingexamples of stabilizing additives include buffer salts, gum acacia,gelatin, methyl cellulose, polyethylene glycol, and polylysine. Thepreferred stabilizing agents are gum acacia, gelatin, and methylcellulose.

The amount of active agent which may be encapsulated by the microsphereis dependent upon a number of factors which include the concentration ofagent in the encapsulating solution as well as the affinity of the agentfor the carrier. The concentration of the active agent in the finalformulation also will vary depending on the required dosage oftreatment. When necessary, the exact concentration can be determined by,for example, reverse phase HPLC analysis.

When the present compositions are in microsphere form, the particle sizeof the microsphere can also aid in providing efficient delivery of theactive agent to the target. Typically, microspheres of the presentinvention will have a diameter of less than 10 μm, preferably in therange of from about 0.1 μm to about 10 μm, and most preferably in therange of from 0.2 μm to about 10 μm. The size of the microspherescontaining an active agent can be controlled by manipulating a varietyof physical or chemical parameters, such as the pH, osmolarity, ionicstrength of the encapsulating solution, or size of the ions in solution,and/or by the choice of the precipitator used in the microsphere formingand loading process.

For example, in the GI tract it is often desirable to use microsphereswhich are sufficiently small to deliver effectively the active agent atthe targeted area within the gastrointestinal tract. Small microspherescan also be administered parenterally by suspending the spheres in anappropriate carrier fluid (e.g. isotonic solution) and injecting thesolution directly into the circulatory system, intramuscularly, orsubcutaneously. The mode of administration of the delivery compositionswill vary, of course, depending upon the requirement of the active agentadministered. It has been noted that large amino acid microspheres(greater than 50 μm) tend to be less effective as oral delivery systems.

The delivery compositions of the present invention may also include oneor more enzyme inhibitors. Such enzyme inhibitors include, but are notlimited to, compounds such as actinonin or epiactinonin and derivativesthereof. These compounds have the formulas below:

Derivatives of these compounds are disclosed in U.S. Pat. No. 5,206,384.Actinonin derivatives have the formula:

wherein R¹² is sulfoxymethyl or carboxyl or a substituted carboxy groupselected from carboxamide, hydroxyaminocarbonyl and alkoxycarbonylgroups; and R¹³ is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group.Other enzyme inhibitors include, but are not limited to, aprotinin(Trasylol) and Bowman-Birk inhibitor.

The delivery compositions of the present invention may be formulatedinto dosage units by the addition of one or more excipient(s),diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s),or dosing vehicle(s). Preferred dosage unit forms are oral dosage unitforms. Most preferred dosage unit forms include, but not limited to,tablets, capsules, or liquids. The dosage unit forms can includebiologically, pharmacologically, or therapeutically effective amounts ofthe active agent or can include less than such an amount if multipledosage unit forms are to be used to administer a total dosage of theactive agent. Dosage unit forms are prepared by methods conventional inthe art.

The compositions of the subject invention are useful for administeringbiologically active agents to any animals such as birds; mammals, suchas primates and particularly humans; and insects. The system isparticularly advantageous for delivering chemical or biologically activeagents which would otherwise be destroyed or rendered less effective byconditions encountered before the microsphere reaches its target zone(i.e. the area in which the active agent of the delivery composition areto be released) and within the body of the animal to which they areadministered. Particularly, the compositions of the present inventionare useful in orally administering active agents, especially those whichare not ordinarily orally deliverable.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation.

Reagents were purchased from Sigma Chemical Co.—St. Louis, Mo., and wereused without further purification. Flash column chromatography wasperformed on Silica gel 40 mm, obtained from J. T. Baker—Co. NMR spectrawere recorded on Varian EM-390, VXR-300, or QE-300 instruments and wererun with chemical shifts given in pats per million downfield from aninternal tetramethylsilane or sodium 3-(trimethylsilyl)-propionatestandard. Mass spectra were obtained on a Kratos MS 80RFA or a Finnigan4516 MS instrument. All optical rotations were performed at 589 nm (theNa D-line) at 22° C. on a Perkin-Elmer 241 polarimeter, with “c”expressed as g of compound per 100 ml of solvent. Melting points areuncorrected.

EXAMPLE 1 Diketopiperazine of L-Glutamic Acid

NαBOC-γ-benzyl-L-glutamic acid (NBGA) (6.0 g, 17.8 mmol) and N-hydroxysuccinimide (2.25 g, 19.6 mmol) were dissolved in anhydroustetrahydrofuran (THF) (150 mL). The solution was cooled to 0° C. in anice bath and dicyclohexylcarbodimide (DCC) (4.04 g, 19.6 mmol) dissolvedin 40 mL anhydrous THF was added dropwise over 30 minutes. The ice bathwas removed. The solution was allowed to warm to room temperature andwas stirred overnight. The reaction was monitored by thin layerchromatography (TLC) (20% EtOH/CHCl₃).

When the reaction was completed, the solution was filtered and thefiltrate was concentrated to provide crude N-hydroxy succinimide (NHS)ester of NαBOC-γ-benzyl-L-glutamic acid as a viscous semi-solid (8.7 g).

Trifluoroacetic acid (TFA, 1.3 mL) was added dropwise to a portion ofthis NHS ester (0.50 g, 1.02 mmol) at 0° C. The solution was slowlyallowed to warm to room temperature and was stirred overnight.

The volatile material was removed at reduced pressure, and a crudeyellow solid (0.85 g) was recrystallized from EtOAc to provide purediketopiperazine L-glutamic acid dibenzyl ester (0.11 g, 50%).

Properties of the diketopiperazine are listed below.

m.p. 275-277° C.

¹H NMR (d₆-DMSO): δ 8.26 (s,2H,NH), 7.46 (s, 10H, aromatic), 5.16 (s,4H, CH₂), 3.98 (t, 2H, CH), 2.58 (m, 4H, CH₂), 2.06 (m, 4H, CH₂).Analysis: Calc. for C₂₄H₂₆N₂O₆: C 66.74, H 5.98, N 6.39: Found: C 65.73,H 6.03, N 6.35. Mass spectrum: Theoretical: 438.18; Found: 439 (M + 1).Optical rotation: [α]_(D) −23.4° (c = 1, dioxane).

EXAMPLE 2 Diketopiperazine of L-Glutamic Acid

The diketopiperazine of L-glutamic acid dibenzyl ester was preparedaccording to the method of Example 1 (0.90 g, 2.05 mmol, 4.1 mequiv.)and was dissolved in a mixture of EtOAc/MeOH (6:1, 470 ml). Pd—C (0.20g) catalyst was added. The black suspension was degassed three times,and hydrogen gas was introduced. The reaction was monitored by TLC (30%EtOH/CHCl₃).

The catalyst was filtered off, and the resultant diacid precipitate waswashed five times with boiling MeOH and EtOAc to dissolve the diacid.The filtrate was concentrated to provide the diketopiperazine ofL-glutamic acid as a white solid (0.53 g, 100%).

Properties of the diketopiperazine are listed below:

m.p. 234-236° C.

¹H NMR (d₇-DMF): δ 4.00 (t, 2H, CH), 2.49 (m, 4H, CH₂), 2.10 (m, 4H,CH₂). Analysis: Calc. for: C₁₀H₁₄N₂O₆: C 46.51; H 5.46; N 10.85: Found:C 46.72; H 5.50; N 10.82. High resolution mass spectrum: Theoretical:259.0930 (M + H); Found: 259.033 (M + H). Optical rotation: [α]_(D) −52°(c = 1, DMSO).

EXAMPLE 3 Diketopiperazine of L-Aspartic Acid

The method of Example 1 was followed, substitutingβ-benzyl-Nα-BOC-L-aspartic acid (24.0 g, 74.2 mmol) for the NBGA, 9.40 g(81.7 mmol) of the NHS, and 16.85 g (81.7 mmol) of the DDC in anhydrousTHF to provide 37.13 g of crude NHS ester.

This NHS ester (37.13 g) was reacted with TFA (85 ml) at 0° C. to yielda crude TFA salt. The salt was neutralized in dry dimethylformamide(DMF) (100 mL) and pyridine (3.5 L) at 0° C. Recrystallization fromEtOAc provided the diketopiperazine of L-aspartic acid dibenzyl ester asa white solid (7.13 g, 47%) m.p. 157° C.

Properties of the diketopiperazine are listed below.

¹H NMR (CDCl₃) δ 7.31 (s, 10H, aromatic) 6.72 (s, 2H, NH), 5.12 (s, 4H,CH₂), 4.35 (m, 2H, CH), 3.00 (m, 4H, CH₂). Analysis: Calc. forC₂₂H₂₂N₂O₆: C 64.38; H 5.40; N 6.83: Found: C 64.27; H 5.39; N 6.79.High resolution mass spectrum: Theoretical: 410.1478: Found: 410.1503.Optical rotation: [α]_(D) −69.50′ (c = 1, CHCL₃).

EXAMPLE 4 Diketopiperazine of L-Aspartic Acid

The diketopiperazine of L-aspartic acid dibenzyl ester (6.15 g, 15 mmol,30 mequiv.) was prepared according to the method of Example 3 and wasdissolved in MeOH (250 mL). Pd—C (0.90 g) catalyst was added. The blacksuspension was degassed three times, and hydrogen gas introduced. Thereaction was monitored by TLC (30% EtOH/CHCl₃).

The catalyst was filtered off, and resultant diacid precipitate waswashed five times with boiling MeOH to dissolve the diacid. The filtratewas concentrated to provide a white solid which was rinsed with MeOH anddried to provide the diketopiperazine of L-aspartic acid as a whitesolid (2.78 g, 80%).

Properties of the diketopiperazine are listed below.

m.p. 254-255° C.

¹H NMR (CDCl₃-d₆ DMSO, 1:1 by vol) δ 7.80 (s, 2H, NH), 4.20 (t, 2H, CH),2.82 (D, 4H, CH₂). Analysis: Calc. for C₈H₁₀N₂O₆: C 41.75; H 4.38; N12.17: Found: C 41.82; H 4.39; N 12.09. Optical rotation: [α]_(D) −37°(c = 1, DMSO).

EXAMPLES 5-8

The diketopiperazines prepared according to the methods of Examples 1-4(0.1 mmol) are dissolved in 0.1 ml of aqueous Li₂CO₃ (1M) deionizedwater to provide a clear solution of the lithium salt. 50 μl of this 1Msolution are mixed with 50 μl of 0.86M citric acid. The mixture isshaken to yield a white suspension. Microspheric examination of thesuspension reveals the presence of tiny spheres which move randomlythroughout the field of inspection. Spheres ranging in size up to about10μ are observed.

EXAMPLES 9-12 Preparation of Diketopiperazine Microspheres ContainingEncapsulated Salmon Calcitonin

Diketopiperazines prepared according to the methods of Examples 1-4 aredissolved at 40° C. in distilled water (640 μL) with 100 ml of Tris basetris(hydroxymethylamine) in distilled water, to prepare a solutionhaving a carrier concentration of 50 mg/ml. Water is added to bring thetotal volume to 4.0 ml. The sample has a carrier concentration of 200mg/mL. Salmon calcitonin (6 μg) and 2M citric acid are added to thesolution. The total salmon calcitonin concentration is 1.5 μg/mL.Microspheres containing salmon calcitonin are observed.

EXAMPLES 13-16 In Vivo Evaluation of Calcitonin Preparations in Rats

Six fasted rats are anesthetized. The rats are administered, by oralgavage, diketopiperazine/calcitonin compositions containing 1.5 μg ofcalcitonin/ml prepared by the methods of Examples 9-12. Each rat isadministered a dosage of 10 μg/kg. The amount of diketopiperazine in thedosage is 300 mg/kg.

Blood samples are collected serially from the tail artery. Serum calciumis determined by testing with a Demand™ Calcium Kit (Sigma ChemicalCompany—St. Louis, Mo.).

COMPARATIVE EXAMPLE 13A In Vivo Evaluation of Calcitonin Preparations inRats

A second group of rats is administered, by oral gavage, 10 μg/kg ofsalmon calcitonin without any carrier.

EXAMPLES 17-20 In Vivo Evaluation of Interferon Preparations in Rats

A dosing preparation is prepared containing interferon α2b anddiketopiperazine carriers prepared according to the methods of Examples1-4 in a Trizma® hydrochloride buffer solution (Tris-HCl) at a pH ofabout 7-8.

The samples containing the interferon α2b and carrier are administeredby oral gavage, to five rats. The dose is 1000 μg/kg. The amount ofcarrier is 800 mg/kg. Delivery is evaluated by using an ELISA assay(BioScience Int.'l.—Camarillo, Calif.) for human interferon α.

EXAMPLES 21-24 Toxicity Studies

Male mice (BALB/c) are fed a dose of 1 g/kg of the diketopiperazineprepared according to the methods of Examples 1-4 per day for five (5)days. The compound shows neither acute nor chronic toxicity (over the 5day period), and no unusual behavior is observed.

EXAMPLE 25-28 In vitro Enzyme Kinetics of Pancreatin Digestion of SalmonCalcitonin

The following solutions are prepared:

Salmon calcitonin (sCt), 10 mg/mil in 0.085 N citric acid; potassiumphosphate (monobasic), 7 mg/ml (titrated to pH 7 with 1 N NaOH);pancreatic, 20 mg/ml in potassium phosphate solution; diketopiperazinecarriers are prepared by dissolving carriers prepared according to themethods of Examples 1-4 in potassium phosphate solution, titrating to pH7.2±0.1 (1 N NaOH or HCl as needed), heating to 37° C., stirring, andfiltering through 0.2μ syringe filter.

Eight 1.7 ml eppendorf tubes are prepared. Two ml of the carriersolution are placed in several 5 ml stoppered tubes. Two ml of potassiumphosphate solution are placed in control tubes (5 ml). 100 μl of sCTstock solution are added to each 5 ml tube. The solutions are vortexed,and a 100 μl aliquot of each tube is transferred to the first eppendorftube in each set (baseline). The eppendorf tubes are immediately cooledto −78° C. in a dry ice/acetone bath and stored for analysis at a latertime. 100 μl of pancreatin stock solution are added to each tube. Thetubes are vortexed. 100 μl of the solution are transferred to a secondeppendorf tube and are frozen. The 5 ml tubes with the reagents areplaced in a 37° C. water bath for one hour. Samples are obtained at thefollowing times 0 min. (baseline), 0.1 min., 1 min., 5 min., 10 min., 15min., 30 min., and 60 min. Samples are kept at −70° C. until ready forassay.

The samples are assayed using HPLC to determine the amount of calcitoninremaining. The conditions are as follows: Column: RANIN C4 3 cm × 4.6mm, 10 μm particle size, 300 Å pore size (Solvent) Mobile Phase A: 10%CH₃CN/90% H₂O in 20 mM potassium phosphate buffer at pH 7 Mobile PhaseB: 60% CH₃CN/40% H₂O in 20 mM potassium phosphate buffer at pH 7 Pump:Hitachi L-6200 Intelligent Pump Linear Gradient: STEP TIME (min) A B 1 070%  30% 2 7 40%  60% 3 7.1 — 100% 4 8.0 — 100% 5 8.1 70%  30% FlowRate: 2.5 ml/min

Step 1-2 is a linear gradient from 70% A/30% B to 40% A/60% B. Steps 2-3is a direct charge to 100% B for 0.9 min followed by a direct charge to70% A/30% B at 8.1 min.

Detector: UV 220 nm.

COMPARATIVE EXAMPLE 25A

The procedure of Examples 25-28 is followed omitting the addition ofcarrier solution to the eppendorf tubes.

All patents, applications, test methods, and publications mentionedherein are hereby incorporated by references.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed disclosure. Allsuch modifications are within the full intended scope of the appendedclaims.

1. A delivery composition comprising: (a) an active agent; and (b) atleast one diketopiperazine selected from the group consisting of: (i) amono-C-substituted diketopiperazine and (ii) a di-C-substituteddiketopiperazine.
 2. A delivery composition as defined in claim 1,comprising a microsphere.
 3. A delivery composition as defined in claim2, wherein said microsphere comprises a microcapsule.
 4. A deliverycomposition as defined in claim 2, wherein said microsphere has adiameter of less than about 10 μm.
 5. A delivery composition as definedin claim 1, wherein said active agent comprises a fragrance.
 6. Adelivery composition as defined in claim 1, wherein said active agentcomprises a biologically active agent.
 7. A delivery composition asdefined in claim 6, wherein said biologically active agent is selectedfrom the group consisting of a peptide, a mucopolysaccharide, acarbohydrate, a lipid, a pesticide, or any combination thereof.
 8. Thedelivery composition as defined in claim 7, wherein saidbiologically-active agent is selected from the group consisting of humangrowth hormone, bovine growth hormone, growth hormone-releasing hormone,an interferon, interleukin-II, insulin, heparin, calcitonin,erythropoietin, atrial naturetic factor, an antigen, a monoclonalantibody, somatostatin, adrenocorticotropin, gonadotropin releasinghormone, oxytocin, vasopressin, cromolyn sodium, vancomycin,desferrioxamine (DFO), or any combination of any of the foregoing.
 9. Adelivery composition as defined in claim 8, wherein saidbiologically-active agent is selected from the group consisting of aninterferon, interleukin-II, insulin, heparin, calcitonin, oxytocin,vasopressin, cromolyn sodium, vancomycin, DFO, or any combination of anyof the foregoing.
 10. A delivery composition as defined in claim 1,wherein said diketopiperazine is derived from two α-amino acids.
 11. Adelivery composition as defined in claim 10, wherein said two α-aminoacids from which said diketopiperazine is derived are independentlyselected from the group consisting of glutamic acid, aspartic acid,tyrosine, phenylalanine, and optical isomers thereof.
 12. A deliverycomposition as defined in claim 10, wherein said two α-amino acids fromwhich said diketopiperazine is derived are the same.
 13. A deliverycomposition as defined in claim 10, wherein said diketopiperazine isprepared by the thermal condensation of said two α-amino acids fromwhich said diketopiperazine is derived.
 14. A delivery composition asdefined in claim 1, further comprising (c) at least one enzymeinhibitor.
 15. A pharmacological composition comprising: (a) at leastone pharmacologically active agent; and (b) at least onediketopiperazine selected from the group consisting of: (i) amono-C-substituted diketopiperazine and (ii) a di-C-substituteddiketopiperazine.
 16. A dosage unit form comprising: (A) a deliverycomposition as defined in claim 1; and (B) (a) an excipient, (b) adiluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) acolorant, (g) a dosing vehicle, or (h) any combination thereof.
 17. Adosage unit form as defined in claim 15, comprising an oral dosage unitform.
 18. A method for administering a biologically active agent to ananimal in need of such agent, said method comprising administeringorally to said animal, a composition as defined in claim
 1. 19. A methodfor preparing microspheres containing an active agent, said methodcomprising: (A) solubilizing, in a solvent, at least onediketopiperazine selected from the group consisting of: (i) amono-C-substituted diketopiperazine and (ii) a di-N-substituteddiketopiperazine. to yield a diketopiperazine solution; and (B)contacting said diketopiperazine solution with said active agent and aprecipitator solution in which said diketopiperazine is insoluble.
 20. Amethod as defined in claim 19, wherein said diketopiperazine solutionhas a pH within a first range and said precipitator solution has a pHwithin a second range, said first range being different than said secondrange.